Lecture 22: Recombinant DNA, DNA-Sequencing, and Genomics

A scientist wishes to produce a mammalian protein in E. coli. The protein is a glycoprotein with a molecular weight of 40,000. Approximately 20% of its mass is polysaccharide. The isolated protein is usually phosphorylated and contains three disulfide bonds. The cloned gene contains no introns. (a) What sequences or sites will be required in the vector to get this gene regulated, transcribed, and translated in E. coli? (b) List two problems in E. coli that might arise in producing a protein identical to that isolated from mammalian cells and describe each problem in no more than two sentences.

(a) The cloned gene must be preceded by a good E. coli promoter and its associated operator and by a ribosome-binding (Shine-Dalgarno) sequence. The other end of the gene should have a transcription terminator sequence. (b) Potential problems are that (1) E. coli enzymes may not glycosylate the protein, which may affect its folding and activity, and (2) the protein kinases that phosphorylate the protein in mammalian cells are probably absent in E. coli; therefore, the engineered protein will not be phosphorylated.

Explain how each of the following is used in cloning in a plasmid: (a) [2 points] antibiotic resistance genes (You must state two different reasons for full credit) (b) [1 point] origin of replication: (c) [1 point] polylinker region:

(a) [2 points] antibiotic resistance genes (You must state two different reasons for full credit) Antibiotic resistance allows a researcher to select for a bacterial cell clone that carries the plasmid; loss of an antibiotic marker in a strain known to contain the plasmid can be used to infer the presence of a cloned DNA segment that interrupts the antibiotic resistance gene. (b) [1 point] origin of replication: An origin of replication assures that the plasmid will replicate autonomously in the bacterium. (c) [1 point] polylinker region: Polylinkers have cut sites for a variety of restriction enzymes, allowing insertion of DNA fragments produced with any of them.

A plasmid that encodes resistance to ampicillin and tetracycline is digested with the restriction endonuclease PstI, which cuts the plasmid at a single site in the ampicillin-resistance gene. The cut plasmid DNA is then ligated with a PstI digest of human DNA and introduced into E. coli cells. (a) What antibiotic would you put in an agar plate to ensure that the cells of a bacterial colony contain the plasmid? (b) What will the antibiotic-resistance phenotypes be of the bacteria growing on the plate? (In your answer, use tetR to refer to tetracycline resistance, tetS to refer to tetracycline sensitivity, ampR to refer to ampicillin resistance and ampS to refer to ampicillin sensitivity.) (c) Which phenotype will indicate the presence of plasmids that contain human DNA fragments? Provide a brief explanation.

(a) tetracycline (b) tetR ampR, tetR ampS (c) tetR ampS This phenotype indicates that the gene for ampicillin resistance has been interrupted by the insertion of a human DNA fragment

What are the steps of PCR?

1) Heat to denature (separate) strands 2) Anneal primers 3) Synthesize DNA copy with heat-stable DNA polymerase 4) Repeat steps 1-3

The diagram below represents a hypothetical operon in the bacterium E. coli. The operon consists of two structural genes (A and B) that code for the enzymes A-ase and B-ase, respectively, and also includes P (promoter) and O (operator) regions as shown. | P | O | A | B | When a certain compound (X) is added to the growth medium of E. coli, the separate enzymes A-ase and B-ase are both synthesized at a 50-fold higher rate than in the absence of X (which has a molecular weight of about 200). Which one of the following statements is true of such an operon? A) Adding X to the growth medium causes a repressor protein to be released from the O region. B) Adding X to the growth medium causes a repressor protein to bind tightly to the O region. C) Synthesis of the mRNA from this operon is not changed by the addition of compound X. D) The mRNA copied from this operon will be covalently linked to a short piece of DNA at the 5' end. E) Two mRNA molecules are made from this operon, one from gene A the other from gene B.

A) Adding X to the growth medium causes a repressor protein to be released from the O region.

In order to perform PCR, the following reagents must be included: A) DNA fragment, primers flanking the region of interest, dNTPs, DNA Polymerase B) DNA fragment, primers flanking the region of interest, dNTPs, ddNTPS, DNA Polymerase C) DNA fragment, one primer, dNTPs, DNA Polymerase, DNA ligase D) DNA fragment, primers flanking the region of interest, dNTPs, DNA Ligase E) none of the above

A) DNA fragment, primers flanking the region of interest, dNTPs, DNA Polymerase

Polymerase chain reaction (PCR) is a technique that allows researchers to amplify a particular segment of DNA. Which ingredients are required for a successful PCR? A) Taq polymerase B) Dideoxynucleoside triphosphates C) Plutonium D) Primers E) Ca2+ ions

A) Taq polymerase D) Primers

What is the essential difference between a genomic library and a cDNA library?

Ans: A genomic library contains (in principle) all of the sequences present in the chromosome(s), including DNA sequences that are not transcribed. Because a cDNA library is made as a DNA copy of mRNA, it contains only those DNA sequences that are expressed in the cell.

The E. coli recombinant plasmid pBR322 has been widely utilized in genetic engineering experiments. pBR322 has all of the following features except: A) a number of conveniently located recognition sites for restriction enzymes. B) a number of palindromic sequences near the EcoRI site, which permit the plasmid to assume a conformation that protects newly inserted DNA from nuclease degradation. C) a replication origin, which permits it to replicate autonomously. D) resistance to two different antibiotics, which permits rapid screening for recombinant plasmids containing foreign DNA. E) small overall size, which facilitates entry of the plasmid into host cells by transformation.

B) a number of palindromic sequences near the EcoRI site, which permit the plasmid to assume a conformation that protects newly inserted DNA from nuclease degradation.

Current estimates indicate that ________ % of the human genome is translated into protein. A) less than 0.5 B) roughly 1.5 C) roughly 10 D) roughly 25 E) more than 50

B) roughly 1.5

Transformation is A) a method of cutting DNA at precise locations. B) a method for covalently joining two DNA molecules. C) a procedure for moving DNA from the test tube into a host. D) A mechanism of carrying and replicating segments of DNA within a host organism. E) A method to identify host cells harboring recombinant DNA molecules.

C) a procedure for moving DNA from the test tube into a host.

Restriction enzymes: A) act at the membrane to restrict the passage of certain molecules into the cell. B) are highly specialized ribonucleases that degrade mRNA soon after its synthesis. C) are sequence-specific DNA endonucleases. D) are very specific proteases that cleave peptides at only certain sequences. E) catalyze the addition of a certain amino acid to a specific tRNA.

C) are sequence-specific DNA endonucleases.

Restriction enzymes: A) act at the membrane to restrict the passage of certain molecules into the cell. B) are highly specialized ribonucleases that degrade mRNA soon after its synthesis. C) are sequence-specific DNA endonucleases. D) are very specific proteases that cleave peptides at only certain sequences. E) catalyze the addition of a certain amino acid to a specific tRNA. Circle the

C) are sequence-specific DNA endonucleases.

Certain restriction enzymes produce cohesive (sticky) ends. This means that they: A) cut both DNA strands at the same base pair. B) cut in regions of high GC content, leaving ends that can form more hydrogen bonds than ends of high AT content. C) make a staggered double-strand cut, leaving ends with a few nucleotides of single-stranded DNA protruding. D) make ends that can anneal to cohesive ends generated by any other restriction enzyme. E) stick tightly to the ends of the DNA they have cut.

C) make a staggered double-strand cut, leaving ends with a few nucleotides of single-stranded DNA protruding.

A molecule that specifically recognizes the desired piece of DNA in a large library is known as a A) vector. B) plasmid. C) probe. D) BAC. E) sensor

C) probe.

In a blue/white screen, A) the blue colonies represent cells transformed with cloning vectors which contain inserts. B) the presence of blue dye identifies cells containing a disrupted α-galactosidase gene. C) the white colonies represent cells transformed with recombinants. D) the foreign DNA is inserted into a chloramphenicol resistance gene E) All of the above

C) the white colonies represent cells transformed with recombinants.

See Exam 3 2011 question 26, it's like the other problem with the type II sequences

Can you read basically, cut them properly.

Sequencing of the human genome revealed that ____ % of the genome represent protein-coding genes. A) 30 B) 55 C) 5 D) 1.5 E) 15

D) 1.5

The PCR reaction mixture does not include: A) oligonucleotide primer(s). B) all four deoxynucleoside triphosphates. C) DNA containing the sequence to be amplified. D) DNA ligase. E) heat-stable DNA polymerase

D) DNA ligase.

In order to amplify Gene X shown below using PCR, to what region and strand would the two oligonucleotide primers anneal? 5' ------------------------------------------ 3' Region A-------Gene X--------Region B 3' ------------------------------------------ 5' A) Region A and B of the top strand. B) Region A of both strands. C) Region A of the top strand and region B of the bottom strand. D) Region A of the bottom strand and region B of the top strand. E) None of the above.

D) Region A of the bottom strand and region B of the top strand.

Which of the following statements about type II restriction enzymes is false? A) Many make staggered (off-center) cuts within their recognition sequences. B) Some cut DNA to generate blunt ends. C) They are part of a bacterial defense system in which foreign DNA is cleaved. D) They cleave and ligate DNA. E) They cleave DNA only at recognition sequences specific to a given restriction enzyme.

D) They cleave and ligate DNA.

A convenient cloning vector with which to introduce foreign DNA into E. coli is a(n): A) yeast transposable element. B) E. coli chromosome. C) messenger RNA. D) plasmid. E) yeast "ARS" sequence.

D) plasmid.

Which of the following statements regarding plasmid cloning vectors is correct? A) Circular plasmids do not require an origin of replication to be propagated in E. coli. B) Foreign DNA fragments up to 400,000 base pairs can be cloned in a typical plasmid. C) Plasmids do not need to contain genes that confer resistance to antibiotics. D) Plasmid vectors must carry promoters for inserted gene fragments. E) Foreign DNA fragments up to 15,000 base pairs can be cloned in a typical plasmid.

E) Foreign DNA fragments up to 15,000 base pairs can be cloned in a typical plasmid.

The technique known as two hybrid analysis for detecting interacting gene products depend on: A) activation of DNA polymerase by the nearby binding of hybridizing protein complexes. B) direct binding of a Gal4p activation domain to a DNA sequence in the promoter region. C) an intact transactivator protein fused to only one of two interacting proteins D) hybridization of DNA segments corresponding to the two genes being examined. E) stimulation of transcription by interaction of two Gal4p domains via fused protein sequences.

E) stimulation of transcription by interaction of two Gal4p domains via fused protein sequences.

Name one enzyme that is always used to make a cDNA library, but is generally not used to make a genomic DNA library. Describe its function in one sentence.

Name of Enzyme: Reverse Transcriptase . Function: Reverse transcriptase is used to make first a single-stranded DNA complementary to mRNA, then a double-stranded DNA

What sequences are required in an expression vector (for use with E. coli) that are not essential in a cloning plasmid?

Regulated expression of the cloned gene requires: (1) a bacterial promoter and (2) its associated operator; (3) a transcription termination sequence; and (4) a ribosomal binding site.

A) Why must the DNA polymerase used in PCR be heat-stable?

The PCR involves repeated heating of the reaction mixture (to denature the double-stranded DNA) and cooling (to allow hybridization of DNA with oligonucleotide primers). A heat-sensitive enzyme would be denatured by this procedure.

Shown at the right are the recognition sequences for several type II restriction endonucleases: BamHi: 5'-G^GATCC-3' 3'-CCTAG^G-5' EcoRI: 5'-G^AATTC-3' 3'-CTTAA^G-5' PvuII: 5'-CAG^CTG-3' 3'-GTC^GAC-5' a) Draw the structure of one end of a linear DNA fragment that was produced by a BamHI restriction digest. (Label all 5ʹ′ and 3ʹ′ ends and include any sequences remaining from the BamHI recognition sequence.) b) Draw the structure resulting from the reaction of the end sequence in (a) with DNA polymerase and the four deoxynucleoside triphosphates. c) Draw the structure of one end of a linear DNA fragment that was produced by a PvuII restriction digest. (Label all 5ʹ′ and 3ʹ′ ends, and include any sequences remaining from the PvuII recognition sequence.) d) Draw the structure of the junction produced if an end with the structure in (b) is ligated to an end with the structure in (c). (Label all 5ʹ′ and 3ʹ′ ends.)

a) Draw the structure of one end of a linear DNA fragment that was produced by a BamHI restriction digest. (Label all 5ʹ′ and 3ʹ′ ends and include any sequences remaining from the BamHI recognition sequence.) 5ʹ′-G-3ʹ′ and 3ʹ′-CCTAG-5ʹ′ or 5ʹ′-GATCC-3ʹ′ and 3ʹ′-G-5ʹ′ b) Draw the structure resulting from the reaction of the end sequence in (a) with DNA polymerase and the four deoxynucleoside triphosphates. 5ʹ′-GGATC-3ʹ′ and 3ʹ′-CCTAG-5ʹ′ or 5ʹ′-GATCC-3ʹ′ and 3ʹ′-CTAGG-5ʹ′ c) Draw the structure of one end of a linear DNA fragment that was produced by a PvuII restriction digest. (Label all 5ʹ′ and 3ʹ′ ends, and include any sequences remaining from the PvuII recognition sequence.) 5ʹ′-CAG-3ʹ′ and 3ʹ′-GTC-5ʹ′ or 5ʹ′-CTG-3ʹ′ and 3ʹ′-GAC-5ʹ′ d) Draw the structure of the junction produced if an end with the structure in (b) is ligated to an end with the structure in (c). (Label all 5ʹ′ and 3ʹ′ ends.) 5ʹ′-GGATCCTG-3ʹ′ and 3ʹ′-CCTAGGAC-5ʹ′ or 5ʹ′-CAGGATCC-3ʹ′ and 3ʹ′-GTCCTAGG-5ʹ′

Shown below is a diagram of the plasmid pBR322: Match each feature of the plasmid pBR322 (at the left) with the appropriate description presented (at the right). ampR sequence ori sequence tetR sequence BamHI sequence EcoRI sequence a) permits selection of bacteria containing the plasmid b) a sequence required for packaging recombinant plasmids into bacteriophage c) origin of replication d) restriction endonuclease recognition sequence e) cleavage of the plasmid here does not affect antibiotic resistance f) insertion of foreign DNA here permits selection of bacteria containing recombinant plasmids

a, f ampR sequence c ori sequence a, f tetR sequence d, f BamHI sequence d, e EcoRI sequence a) permits selection of bacteria containing the plasmid b) a sequence required for packaging recombinant plasmids into bacteriophage c) origin of replication d) restriction endonuclease recognition sequence e) cleavage of the plasmid here does not affect antibiotic resistance f) insertion of foreign DNA here permits selection of bacteria containing recombinant plasmids

Match each vector with the appropriate method used for its introduction into host cells: (a) Electroporation (b) Transformation (c) Infection Plasmid Bacteriophage λ BAC (Bacterial Artificial Chromosome)

b Plasmid c Bacteriophage λ a BAC (Bacterial Artificial Chromosome) (a) Electroporation (b) Transformation (c) Infection

Which of the following partial amino acid sequences from a protein whose gene you wish to clone would be most useful in designing an oligonucleotide probe to screen a cDNA library? (Circle the correct answer.) a. Met-Leu-Arg-Leu b. Met-Trp-Cys-Trp Explain your choice (in less than 20 words).

b. Met-Trp-Cys-Trp The best probe has minimal codon degeneracy.

E. coli cells are placed in a growth medium containing lactose. Indicate how the following circumstances would affect the expression of the lactose operon (write the word increase, decrease, or no change in the spaces provided). Addition of high levels of glucose A Lac repressor mutation that prevents dissociation of Lac repressor from the promoter A mutation that inactivates b-galactosidase A mutation that inactivates galactoside permease A mutation that prevents binding of CRP to its binding site near the lac promotor

decrease decrease decrease decrease decrease

Explain briefly the properties of the plasmid pBR322 that make it so convenient as a vector for cloning fragments of foreign DNA.

pBR322 has two antibiotic resistance markers, so that its presence in a bacterium can be detected, bacteria that carry it can be selected, and insertion of cloned DNA into one of the resistance markers can be detected by loss of antibiotic resistance. The plasmid also has an origin of replication, so that it replicates autonomously in E. coli. Several convenient restriction sites allow easy insertion of restricted DNA fragments. Its small size facilitates its entry into E. coli by standard transformation protocols.

For each of the five basic procedures that are necessary for DNA (or molecular) cloning, fill in the blank: A) A method for cutting DNA at precise locations, which relies on _____________________________________. B) An enzyme for covalently joining two DNA molecules: ______________. C) Procedures for moving recombinant DNA from the test tube into a host. Name two: __________________________________. D) A vehicle for carrying and replicating segments of DNA within a host organism: ________________________________. E) A method (name one) to select and identify those host cells harboring desired recombinant DNA molecules: __________________.

restriction endonucleases DNA ligase Transformation, infection, electroporation vector antibiotic resistance gene, blue/white selection